Methods for predicting and treating myocardial damage

ABSTRACT

A method for predicting myocardial damage in a subject having or at risk of cardiac disease includes determining a level of apolipoprotein AI (ApoAI) and a level of Coenzyme Q 10  (CoQ 10 ) in the subject and comparing the determined levels of ApoAI and CoQ 10  to control levels.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.61/384,969, filed, Sep. 21, 2010, the subject matter of which isincorporated herein by reference in their entirety.

TECHNICAL FIELD

This application relates generally to methods and kits for predictingmyocardial damage in a subject having or at risk of cardiac disease andto methods for mitigating ischemic damage in a subject having anincreased risk of myocardial damage resulting from cardiac disease.

BACKGROUND OF THE INVENTION

Plasma levels of high-density lipoproteins (HDL) and apolipoprotein AI(ApoAI) are inversely associated with cardiovascular morbidity andmortality. ApoAI is primarily synthesized in the liver and smallintestine, and comprises a single polypeptide of 243 amino acid residueswith a molecular weight of approximately 28,000 Da. ApoAI, throughactivation of lecithin:cholesterol acyltransferase, catalyzes thereaction of cholesterol and phosphatidylcholine to yield cholesterolesterified with a long-chain fatty acid and 2-lysophosphatidylcholine,an important step in reverse cholesterol transport.

The importance of HDL cholesterol as an independent risk factor forcoronary artery disease (CAD) is well known. Novel therapeuticapproaches for administering HDL protein, ApoAI, or ApoAI analogues toalter the development of atherosclerosis have been investigated inanimal models and in humans. In fact, individuals with ApoAI deficiencyand ApoAI-deficient mice fail to form normal HDL particles and, as aresult, are predisposed to premature CAD.

Recent studies have also shown anti-inflammatory properties of ApoAI.For example, the inhibitory activity of ApoAI appears to be specificallydirected to contact-mediated monocyte activation by T-cells throughinhibition of TNF-α and IL1β. In addition to anti-inflammatory andanti-atherogenic function, reduced plasma concentrations of HDL andApoAI have been implicated in the development of Type 2 diabetes.

SUMMARY OF THE INVENTION

An aspect of the application relates to a method for predictingmyocardial damage in a subject having or at risk of cardiac disease. Themethod includes determining a level of apolipoprotein AI (ApoAI) and alevel of CoQ₁₀ in the subject. The method further includes comparing thedetermined levels of ApoAI and a CoQ₁₀ to control levels. A decreasedlevel of ApoAI and a decreased level of CoQ₁₀ compared to control levelsare indicative of the subject having an increased risk of greatermyocardial damage following a myocardial infarction.

Another aspect of the application relates to a method for determiningincreased risk of greater myocardial damage in a subject having or atrisk of cardiac disease. The method includes determining a level ofapolipoprotein AI (ApoAI) and a level of CoQ₁₀ in the subject. Themethod further includes comparing the determined levels of ApoAI and aCoQ₁₀ to control levels. A decreased level of ApoAI and a decreasedlevel of CoQ₁₀ compared to control levels are indicative of the subjecthaving an increased risk of greater myocardial damage following amyocardial infarction.

Another aspect of the application relates to a kit for predictingmyocardial damage in a subject having or at risk of cardiac disease. Thekit includes a first reagent for determining a level of ApoAI in thesubject. The kit also includes a second reagent for determining a levelof CoQ₁₀ in the subject and instructions for predicting myocardialdamage in a subject having or at risk of cardiac disease.

A further aspect of the application relates to a method of mitigatingischemic damage in a subject having an increased risk of myocardialdamage resulting from cardiac disease. The method includes administeringtherapeutically effective amounts of CoQ₁₀ and a hypolipidemic agent toa subject, which has decreased levels of ApoAI and CoQ₁₀ as compared toa control.

Yet another aspect of the application relates to a pharmaceuticalcomposition for mitigating ischemic damage in a subject having anincreased risk of myocardial damage resulting from cardiac disease. Thepharmaceutical composition includes therapeutically effective amounts ofCoQ₁₀ and a hypolipidemic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features of the present invention will becomeapparent to those skilled in the art to which the present inventionrelates upon reading the following description with reference to theaccompanying drawings, in which:

FIG. 1 is a plot comparing infarct size as a percent area at risk (AAR)in wild-type (WT), ApoAI-heterozygote (+/−), and ApoAI-null (−/−) miceafter 30 minutes of ischemia and 3 hours of reperfusion (data representmean+/−SD);

FIGS. 2A-B are a series of images following hydroethidine staining forreactive oxygen species (ROS) production in myocardial tissue from WT(FIG. 2A) and ApoAI-null (FIG. 2B) mice 1 hour after reperfusion. FIG.2C illustrates the mean intensity quantified over 4 animals per group.Twelve random images from within the infarct zone from each animal werequantified (data represent mean±SD);

FIGS. 3A-B are a series charts showing electron transport chainactivity. FIG. 3A is a plot comparing the relative activities of complexI, complex II, and complex III. FIG. 3B is a plot comparing the relativeactivity of NADH cytochrome c reductase (NCR) and succinate cytochrome creductase (SCR); and

FIG. 4 is a plot comparing infarct size as a percentage of AAR in WT andApoAI-null mice given saline or Coenzyme Q10 daily for 3 days prior toischemia induced by 30 minutes of LAD ligation and 3 hours ofreperfusion (data represent mean±SD; n=4-6 per group; *p<0.0001 comparedto strain-matched NT control).

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates). Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thepresent invention pertains. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th Ed., Springer-Verlag: New York,1991, and Lewin, Genes V, Oxford University Press: New York, 1994. Thedefinitions provided herein are to facilitate understanding of certainterms used frequently herein and are not meant to limit the scope of thepresent invention.

As used herein, the term “polypeptide” can refer to an oligopeptide,peptide, polypeptide, or protein sequence, or to a fragment, portion, orsubunit of any of these, and to naturally occurring or syntheticmolecules. The term “polypeptide” can also include amino acids joined toeach other by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain any type of modified amino acids. The term“polypeptide” can also include peptides and polypeptide fragments,motifs and the like, glycosylated polypeptides, and all “mimetic” and“peptidomimetic” polypeptide forms.

As used herein, the term “amino acid” can refer to naturally occurringand synthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. “Aminoacid analogs” can refer to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” can refer tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but function in a mannersimilar to a naturally occurring amino acid.

As used herein, the term “polynucleotide” can refer to oligonucleotides,nucleotides, or to a fragment of any of these, to DNA or RNA (e.g.,mRNA, rRNA, tRNA) of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent a sense orantisense strand, to peptide nucleic acids, or to any DNA-like orRNA-like material, natural or synthetic in origin, including, e.g.,iRNA, ribonucleoproteins (e.g., iRNPs). The term can also encompassnucleic acids, i.e., oligonucleotides, containing known analogues ofnatural nucleotides. Additionally, the term can encompass nucleicacid-like structures with synthetic backbones.

As used herein, the term “pharmaceutically acceptable carrier” caninclude any material, which when combined with a conjugate retains theconjugate's activity and is non-reactive with a subject's immune system.Examples include, but are not limited to, any of the standardpharmaceutical carriers, such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsion, and various types ofwetting agents. Other carriers may also include sterile solutions,tablets including coated tablets, and capsules. Typically, such carrierscontain excipients, such as starch, milk, sugar, certain types of clay,gelatin, stearic acid or salts thereof, magnesium or calcium stearate,talc, vegetable fats or oils, gums, glycols, or other known excipients.Such carriers may also include flavor and color additives or otheringredients. Compositions comprising such carriers are formulated bywell known conventional methods.

As used herein, the term “subject” can refer to any animal, including,but not limited to, humans and non-human animals (e.g., rodents,arthropods, insects, fish (e.g., zebrafish)), non-human primates,ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines,canines, felines, ayes, etc.), which is to be the recipient of aparticular treatment. Typically, the terms “patient” and “subject” areused interchangeably herein in reference to a human subject.

As used herein, the terms “administer” or “administering” can refer tooral administration, administration as a suppository, topical contact,intravenous, intraperitoneal, intramuscular, intralesional, intranasalor subcutaneous administration, or the implantation of a slow-releasedevice e.g., a mini-osmotic pump to a subject. Administration can be byany route, including parenteral and transmucosal (e.g., oral, nasal,vaginal, rectal or transdermal). Parenteral administration can include,e.g., intravenous, intramuscular, intra-arteriole, intradermal,subcutaneous, intraperitoneal, intraventricular, and intracranial. Othermodes of delivery can include, but are not limited to, the use ofliposomal formulations, intravenous infusion, transdermal patches, etc.

As used herein, the terms “cardiac disease,” “cardiac disorder,”“cardiovascular disease”, “cardiovascular disorder,” or “cardiovascularcondition” can refer to any disease or disorder that negatively affectsthe cardiovascular system. The terms can also refer to cardiovascularevents. “Cardiovascular events”, as used herein, can include acutecoronary syndrome, myocardial infarction, myocardial ischemia, chronicstable angina pectoris, unstable angina pectoris, angioplasty, stroke,transient ischemic attack, claudication(s) and vascular occlusion(s).Cardiac diseases and disorders, therefore, can include acute coronarysyndrome, myocardial infarction, myocardial ischemia, chronic stableangina pectoris, unstable angina pectoris, angioplasty, stroke,transient ischemic attack, claudication(s), vascular occlusion(s),arteriosclerosis, left ventricular dysfunction, heart failure, andcardiac hypertrophy.

This application relates to methods for diagnosing, predicting, and/ordetermining an increased risk of myocardial damage in a subject and tomethods for mitigating myocardial damage in a subject having anincreased risk of myocardial damage resulting from cardiac disease. Itwas found that: (1) myocardial infarct (MI) size in apolipoprotein AI(ApoAI)-deficient mice is substantially greater than MI size inwild-type (WT) mice; (2) prophylactic treatment of ApoAI-deficient micewith Coenzyme Q₁₀ (CoQ₁₀) prior to MI leads to a 100% decrease in MIsize; and (3) analyses of mitochondrial function in WT andApoAI-deficient mice suggests that there is a defect in the mitochondriaof ApoAI-deficient mice that leads to decreased flux in the succinatecytochrome c reductase (SCR) pathway secondary to a deficiency in the Qpool.

Based on these discoveries, it was determined that subjects, which havedecreased ApoA1 and CoQ₁₀ levels compared to control subjects haveincreased myocardial damage (e.g., ischemic damage) following myocardialinfarction and that the ApoA1 and CoQ₁₀ levels of a subject can bemeasured to determine or predict if the subject has increased risk ofgreater myocardial damage (e.g., ischemic damage) following myocardialinfarction. The ability to determine a subject as having an increasedrisk of greater myocardial damage, therefore, provides a usefuldiagnostic tool to predict the amount of myocardial damage in a subjectresulting from cardiac disease and to help mitigate or preventmyocardial damage in subjects at risk of cardiovascular disease (e.g.,MI).

Accordingly, an aspect of the application relates a method forpredicting myocardial damage in a subject having or at risk of cardiacdisease. The method includes determining the levels of the cardiacmarkers, apolipoprotein and CoQ₁₀ in a subject.

As used herein, the term “apolipoprotein” can refer to apolipoproteinsknown to those of skill in the art and variants and fragments thereof.Apolipoproteins are proteins that bind to lipids and transport dietaryfats through the bloodstream. Apolipoproteins that may be used ascardiac markers to predict the amount of myocardial damage in a subjectinclude, but are not limited to, Apolipoprotein (Apo) A (e.g., ApoAI,ApoAII, ApoIV and ApoV), ApoB (e.g., ApoB48 and ApoB100), ApoC (e.g.,ApoCI, ApoCII, ApoCIII and ApoCIV), ApoD, ApoE and Apo H. In oneexample, the level of ApoAI (e.g., an ApoAI polypeptide) in a subjectcan be used to determine an increased risk of greater myocardial damagein a subject.

ApoAI is the major protein component of high-density lipoprotein complex(HDL) and chylomicrons secreted from the intestinal enterocyte alsocontain ApoAI, however it is quickly transferred to HDL in a subject'sbloodstream. Accordingly, the application also contemplates that a levelof HDL in a subject can be indicative of a level of ApoAI in thesubject. Therefore, in another example, the levels of HDL and CoQ₁₀ in asubject can be used to determine an increased risk of greater myocardialdamage in a subject.

The levels of the cardiac markers (e.g., ApoAI and CoQ₁₀) can bedetermined by first obtaining one or more biological samples from asubject. In one example, the levels of the cardiac markers ApoAI andCoQ₁₀ can both be determined by first obtaining a single biologicalsample from a subject. In another example, the levels of the cardiacmarkers, ApoAI and CoQ₁₀, can each be determined separately by obtainingtwo or more biological samples from a subject.

The subject can be an apparently healthy subject, a subject at risk fora cardiovascular disease, or a subject known to have cardiovasculardisease. “Apparently healthy”, as used herein, can refer to subjects whohave not previously been diagnosed as having any signs or symptomsindicating the presence of a cardiac disease, a history of a cardiacdisease, or evidence of a cardiac disease. Apparently healthy subjectsmay not otherwise exhibit symptoms of a cardiac disease. In other words,such subjects, if examined by a medical professional, would becharacterized as healthy and free of symptoms of a cardiac disease.

Subjects at risk for a cardiac disease can exhibit any one orcombination of risk factors for cardiovascular disease including, butnot limited to, elevated blood pressure, an abnormal response to astress test, elevated levels of myeloperoxidase, C-reactive protein, lowdensity lipoprotein, cholesterol, or atherosclerotic plaque burden.Techniques for assessing cardiovascular disease risk factors are knownin the art and can include coronary angiography, coronary intravascularultrasound, stress testing (with and without imaging), assessment ofcarotid intimal medial thickening, carotid ultrasound studies with orwithout implementation of techniques of virtual histology, coronaryartery electron beam computer tomography, cardiac computerizedtomography (CT) scan, CT angiography, cardiac magnetic resonanceimaging, and magnetic resonance angiography.

The biological sample can include whole blood samples and samples ofblood fractions, such as serum and plasma. The biological sample may befresh blood, stored blood (e.g., in a blood bank), or a blood fraction.The biological sample may be a blood sample expressly obtained for theassay(s) described herein or, alternatively, a blood sample obtained foranother purpose which can be sub-sampled. In one example, the biologicalsample can comprise whole blood. Whole blood may be obtained from thesubject using standard clinical procedures. In another example, thebiological sample can comprise plasma. Plasma may be obtained from wholeblood samples by centrifugation of anti-coagulated blood. Such processprovides a buffy coat of white cell components and a supernatant of theplasma. In yet another example, the biological sample can compriseserum. Serum may be obtained by centrifugation of whole blood samplesthat have been collected in tubes that are free of anti-coagulant. Theblood may then be permitted to clot prior to centrifugation. Theyellowish-reddish fluid obtained by centrifugation is the serum.

Biological samples can be pretreated as necessary by dilution in anappropriate buffer solution, heparinized, concentrated if desired, orfractionated by any number of methods including, but not limited to,ultracentrifugation, fractionation by fast performance liquidchromatography, precipitation with dextran sulfate, or other knownmethods. Any number of standard aqueous buffer solutions employing oneor a combination of buffers, such as phosphate, Tris, or the like, atphysiological pH can also be used.

In one example, a biological sample including CoQ₁₀ can include serum.In another example, a biological sample can be obtained from a subjectwhere the level of CoQ₁₀ included in the sample reflects the CoQ₁₀tissue level instead of the dietary intake of a subject. For example abiological sample can include cultured skin fibroblasts, muscle biopsiesand blood mononuclear cells.

After obtaining the biological sample from the subject, the levels ofthe cardiac markers (e.g., ApoAI and CoQ10) are determined using any oneor combination of known biochemical assays or techniques. Examples ofbiochemical assays or techniques that can be used to determine the levelof an ApoAI polypeptide, HDL, and/or CoQ₁₀ can include, for example,antibody based assays, such as ELISA and Western blots, massspectroscopy (MS) (e.g., LC/ESI/MS/MS), fluorometric assays andchromatography (e.g., HPLC, affinity column, etc.). It will beappreciated that biochemical assays or techniques may also be used todetermine the level of a cardiac marker comprising a polynucleotide. Forexample, the level of an mRNA encoding an ApoAI polypeptide can bedetermined using Northern blot analysis. Alternatively, the presence orabsence of the gene encoding an ApoAI polypeptide can be determinedusing PCR, for example.

In an embodiment of the application, the level of CoQ₁₀ can bedetermined using high-performance liquid chromatography (HPLC) withelectrochemical detection as described by Tang et al., ClinicalChemistry 2001; 47(2)256-265), which is incorporated herein byreference.

Once the levels of the cardiac markers have been determined, the levelsof the cardiac markers are compared to control levels in order todetermine an increased risk of greater myocardial damage in a subjectfollowing a myocardial infarction. For example, the level of an ApoAIpolypeptide in a biological sample can be determined using ELISA and thelevel of CoQ₁₀ in a biological sample can be determined using HPLC andthen both levels can be compared to control levels or values of ApoAIand CoQ₁₀, respectively. The control levels can be based upon the levelof an ApoAI polypeptide or CoQ₁₀ in a comparable biological sample (orsamples) obtained from a control population (e.g., the generalpopulation) or a select population of subjects. For example, the selectpopulation may be comprised of apparently healthy subjects, subjectsdetermined to have myocardial damage resulting from cardiac disease,subjects determined to have little or no myocardial damage resultingfrom cardiac disease or from subjects at risk for a cardiac disease.

The control levels can be related to the levels used to characterize thelevels of the ApoAI polypeptide and CoQ₁₀ obtained from the subject. Forexample, if the level of the ApoAI polypeptide is an absolute value,such as the units of ApoAI polypeptides per ml of blood, the controllevel can also based upon the units of ApoAI polypeptides per ml ofblood in subjects of the general population or a select population.Similarly, if the level of the ApoAI polypeptide and CoQ₁₀ is arepresentative value, such as an arbitrary unit obtained from an ELISA,the control level can also be based on the representative value.

The control levels can also take a variety of forms. For example, thecontrol levels can be a single cut-off value, such as a median or mean.The control levels can be established based upon comparative groups,such as where the risk in one defined group is double the risk ofanother defined group. The control levels can also be divided equally(or unequally) into groups, such as a low-risk group, a medium-riskgroup, and a high-risk group, or into quadrants, the lowest quadrantbeing subjects with the lowest risk the highest quadrant being subjectswith the highest risk.

Control levels of ApoAI polypeptides and CoQ₁₀ in biological samples,for example, can be obtained (e.g., mean levels, median levels, or“cut-off” levels) by assaying a large sample of subjects in the generalpopulation or a select population and then using a statistical model,such as the predictive value method for selecting a positivity criterionor receiver operator characteristic curve that defines optimumspecificity (highest true negative rate) and sensitivity (highest truepositive rate), as described in Knapp, R. G. and Miller, M. C. (1992):Clinical Epidemiology and Biostatistics, William and Wilkins, HarualPublishing Co. (Malvern, Pa.), which is incorporated herein byreference.

Depending upon the levels or values of the cardiac markers when comparedto the control levels, a determination can be made as to the risk ofgreater myocardial damage in the subject following a myocardialinfarction. In some embodiments, the myocardial damage can be defined asthe ratio of ischemic or infracted myocardial area to total myocardialarea and, as described below, can be expressed as a percentage. In anexample of the method, a reduced or decreased level of an ApoAIpolypeptide in combination with a reduced level of CoQ₁₀ as compared tocontrol value levels may indicate an increased risk of developing agreater amount of myocardial damage. Thus, a subject with a reducedlevel of an ApoAI polypeptide and a reduced level of CoQ₁₀ may have anincreased risk of developing a larger infarct area in the left ventricle(e.g., as a result of MI) as compared to a control subject.Alternatively, a normal or increased level of an ApoAI polypeptide andCoQ₁₀ as compared to a control value level may indicate little or norisk of a subject developing greater or increased myocardial damagefollowing MI.

In another aspect of the application, a kit is provided for diagnosingan increased risk of myocardial damage resulting from cardiac disease ina subject. The kit includes at least one first reagent that specificallydetects and/or determines the level of ApoAI, such as an ApoAIpolypeptide, an ApoAI polypeptide fragment, a polynucleotide encoding anApoAI polypeptide, or a polynucleotide encoding a fragment of an ApoAIpolypeptide in a subject, at least one second reagent that specificallydetects and/or determines the level of CoQ₁₀, in a subject andinstructions for using the kit to determine an increased risk of greatermyocardial damage in a subject following a myocardial infarction.

In an example of the application, a first reagent can detect expressionlevels of an ApoAI polypeptide or fragment thereof via an antibody thatspecifically binds to the ApoAI polypeptide or fragment thereof. Inother example, the first reagent can comprises a nucleic acid probecomplementary to a polynucleotide sequence coding for an ApoAIpolypeptide or fragment thereof. For example, the nucleic acid probe maybe a cDNA or an oligonucleotide immobilized on a substrate surface.

The instructions of the kit can include instructions required by aregulatory agency (e.g., the U.S. Food and Drug Administration) for usein in vitro diagnostic products. For example, the instructions can beapplicable to one or more of an extraction buffer/reagent(s) and arelated protocol, an amplification buffer/reagent(s) and a relatedprotocol, a hybridization buffer/reagent(s) and a related protocol, animmunodetection buffer/reagent(s) and a related protocol, a labelingbuffer/reagent(s) and a related protocol, and/or a control value orvalues (as described above).

This application also relates to a method for mitigating ischemic damagein a subject having an increased risk of a myocardial damage resultingfrom cardiac disease. The method includes administering therapeuticallyeffective amounts of a ubiquinone in combination with a hypolipidemicagent to the subject. In one example, the subject is determined to havean increased risk of a myocardial damage resulting from cardiac diseaseas described herein.

Administration of a ubiquinone “in combination with” or “in conjunctionwith” a hypolipidemic agent includes parallel administration(administration of both the agents to the patient over a period-of time,such as administration on alternate days for one month)co-administration (in which the agents are administered at approximatelythe same time, e.g., within about a few minutes to a few hours of oneanother), and co-formulation (in which the agents are combined orcompounded into a single dosage form suitable for oral or parenteraladministration).

As used herein, the term “therapeutically effective amounts” can referto the amount of a ubiquinone administered to a subject in combinationwith an amount of a hypolipidemic agent that results in lowering oreliminating the risk of ischemic damage in a subject found to have anincreased risk of myocardial damage. A therapeutically effective amountcan also refer to a prophylactically effective amount. As used herein, a“prophylactically effective amount” is an amount of a ubiquinone and ahypolipidemic agent that, when administered to a subject, will have theintended prophylactic effect, e.g., preventing or delaying the onset (orreoccurrence) of cardiac disease or symptoms, or reducing the likelihoodof the onset (or reoccurrence) of cardiac disease or symptoms. The fullprophylactic effect does not necessarily occur by administration of onedose and may occur only after administration of a series of doses. Thus,a prophylactically effective amount may be administered in one or moreadministrations.

The ubiquinone co-administered with a hypolipidemic agent to the subjectcan include one or series of quinones, which are widely distributed inanimals, plants and microorganisms, a ubiquinone mimetic, a ubiquinonevariant, or a ubiquinone fragment. In one example of the presentinvention the ubiquinone co-administered to a subject with ahypolipidemic agent is CoQ₁₀.

CoQ functions as an agent for carrying out oxidation and reductionwithin cells. Its primary site of function is in the terminal electrontransport system where it acts as an electron or hydrogen carrierbetween the flavoproteins (which catalyze the oxidation of succinate andreduced pyridine nucleotides) and the cytochromes. This process iscarried out in the mitochondria of cells of higher organisms. CoQ playsan important role as an antioxidant to neutralize potentially damagingfree radicals created in part by the energy-generating process. Forexample, CoQ₁₀ has antioxidant and membrane stabilizing properties thatserve to prevent cellular damage resulting from normal metabolicprocesses.

The term “hypolipidemic agents” as used herein refers to several classesof pharmaceuticals that are well known to increase ApoA1 levels and/orHDL levels in vivo. In some embodiments, a hypolipidemic agentadministered to the subject in combination with ubiquinone in accordancewith the applicaion can include ApoAI polypeptides, ApoAI mimetics,ApoAI analogs, cholesteryl ester transfer protein (CETP) inhibitors andstatins.

“ApoAI polypeptides” as used herein refers to ApoAI peptide fragmentsand full length proteins. By “ApoAI mimetics” or “mimetics of ApoAI” or“known mimetics of ApoAI” as used in the specification and in theclaims, it is meant mimetics of ApoA1 that can be identified or derivedfrom any reference and that have ApoA1 behavior. These include mimeticsof ApoAI identified in U.S. and foreign patents and publications. Forexample, an ApoA1 mimetic described herein can include any number ofpeptidomimetics of ApoA1 designed to beneficially influence the lipidparameters and/or cholesterol levels in the blood. Accordingly, an ApoA1polypeptide mimetics contemplated herein may include modifiedpolypeptides from the ApoA1 forms and variants including, for example,apolipoprotein A-1 (Brewer et al., (1978)), apolipoprotein A-1 Milano(Weisgraber (1983) J. Biol. Chem. 258: 2508-2513), apolipoprotein A-1Marburg, (Utermann et al., (1982) J. Biol. Chem. 257: 501-507),apolipoprotein A-1 Paris (Bielicki and Oda (2002) Biochemistry 41,2089-2096), proapolipoprotein A-1, or any other mutant form of ApoA1known in the art whether synthetically formed or naturally occurring.

An ApoAI mimetic can also include an ApoAI agonist which mimics thefunction of ApoAI in a subject. An example of an ApoAI agonist includesthe recombinant ApoAI mutant protein referred to as the ‘milano’ mutant.The Milano mutant ApoA1 has an Arginine to Cysteine mutation at aminoacid position 197 (R197c) in the pre-pro-ApoA1 protein amino acidsequence (corresponding to R173c in the mature ApoA1 amino acidsequence). The cysteine in the milano mutant leads to the formation ofan ApoA1 dimer, held together by a disulfide bond, due to the additionalcysteine residue.

Additional ApoA1 mimetics include ApoA1 oxidant resistant mimetics, suchas those describe in U.S. Pat. Appl. No. 20090149390A1, which isincorporated herein by reference. For example, an ApoA1 mimetic for usein a method described herein can include, but is not limited to, anApoA1 mimetic having an amino acid sequence that includes at least aportion of the amino acid sequence of ApoA1 or a mimetic of the ApoA1where at least one tryptophan has been substituted with an oxidationresistant amino acid, (e.g., phenylalanine) in the amino acid sequenceof the ApoA1 mimetic. An ApoA1 mimetic for use in the present inventioncan also include stabilized Apo A1 protein variants such as thosedescribed in U.S. Pat. Appl. No.: 20100222276A1, which is incorporatedherein by reference.

As used herein, the term “statins” or “statin drug” can refer to anycompound or agent capable of substantially inhibiting HMG Co-Areductase. Statins are a family of molecules sharing the capacity tocompetitively inhibit the hepatic enzyme 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase. This enzyme catalyses the rate-limitingstep in the L-mevalonate pathway for cholesterol synthesis.Consequently, statins block cholesterol synthesis. Statins that can beadministered, or co-administered to a subject according to the inventioninclude, Compactin, Atorvastatin, Pravastatin, Lovastatin, Mevinolin,Pravastatin, Fluvastatin, Mevastatin, Visastatin/RosuvastatinVelostatin,Cerivastatin, Simvastatin, Synvinolin, Rivastatin (sodium7-(4-fluorophenyl)-2,6-diisoprop-yl-5-methoxymethylpyridin-3-yl)-3,5-dihy-droxy-6-heptanoate),and Itavastatin/Pitavastatin. In one example, statins are administeredorally from about 1 mg/day to about 40 mg/day.

Additional hypolipidemic agents for use in the compositions and methodsdescribed herein include but are not limited to bile acid sequestrants(resins), ezetimibe, phytosterols, olistat, acipimox CETP inhibitors,squalene synthase inhibitors, AGI-1067 and mipomersin. Clinically, thechoice of an agent will depend on the patient's cholesterol profile,cardiovascular risk, and the liver and kidney functions of the patientevaluated against the balancing of the risks and benefits of thehypolipdemic agent.

A hypolipidemic agent administered to a subject in combination with anubiquinone (e.g., CoQ₁₀) in accordance with the application can alsoinclude an agent that increases HDL-cholesterol in a subject. Forexample, an agent that increases HDL-cholesterol in a subject caninclude niacin and/or fibrates. In one example, pharmacologic niacin(about 1- to about 3-gram/day) can be administered to a subject incombination with CoQ₁₀.

The therapeutically effective amounts of ubiquinone (e.g., CoQ₁₀) and/ora hypolipidemic agent can be administered in an isolated or concentratedform, or as a part of one or more pharmaceutical compositions and/orformulations. In one embodiment, a pharmaceutical composition caninclude ubiquinone and a hypolipidemic agent as the active ingredientand a pharmaceutically acceptable carrier or aqueous medium excipientsuitable for administration and delivery in vivo. Combined therapeuticsare also contemplated, and the same type of underlying pharmaceuticalcompositions may be employed for both single and combined medicaments.For example, a pharmaceutical composition described herein can includeubiquinone and a hypolipidemic agent described above as the activeingredients and a pharmaceutically acceptable excipient suitable foradministration and delivery in vivo.

A pharmaceutical composition described herein can be administered by anyappropriate route, such as percutaneous, parenteral, subcutaneous,intravenous, intraarticular, intrathecal, intramuscular,intraperitoneal, or intradermal injections, or by transdermal, buccal,oromucosal, ocular routes, or via inhalation. The dosage administeredwill be dependent upon the age, health, and weight of the subject, kindof concurrent treatment, if any, frequency of treatment, and the natureof the effect desired. In a subject with both a decreased level of anApoAI polypeptide and a decreased level of CoQ₁₀, for example, atherapeutically effective amount of a pharmaceutical compositioncomprising CoQ₁₀ can be prophylactically administered to prevent ormitigate ischemic damage (e.g., as a result of MI).

In addition to one or more active ingredients (e.g., CoQ₁₀),pharmaceutical compositions can include pharmaceutically acceptablecarriers comprising excipients and auxiliaries that facilitateprocessing of an active ingredients into pharmaceutical preparations.The pharmaceutical preparations of the present invention can bemanufactured in a known manner by, for example, means of conventionalmixing, granulating, dragee-making, dissolving, or lyophilizingprocesses. Thus, pharmaceutical preparations for oral use can beobtained by combining the active agents with solid excipients,optionally grinding the resulting mixture, and processing the mixture ofgranules after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients can include fillers, such as saccharides (e.g.,lactose or sucrose, mannitol or sorbitol), cellulose preparations and/orcalcium phosphates (e.g., tricalcium phosphate or calcium hydrogenphosphate), as well as binders, such as starch paste using, for example,maize starch, wheat starch, rice starch, potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents can be added, such as the above-mentionedstarches, as well as carboxymethyl-starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodiumalginate. Auxiliaries are flow-regulating agents and lubricants, forexample, silica, talc, stearic acid or salts thereof, such as magnesiumstearate, or calcium stearate, and/or polyethylene glycol. Dragee corescan be provided with suitable coatings that, if desired, are resistantto gastric juices. For this purpose, concentrated saccharide solutionscan be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol, and/or titanium dioxide, lacquersolutions, and suitable organic solvents or solvent mixtures.

To produce coatings resistant to gastric juices, solutions of suitablecellulose preparations, such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate can be used. Slow-release andprolonged-release formulations may be used with particular excipients,such as methacrylic acid-ethylacrylate copolymers, methacrylicacid-ethyl acrylate copolymers, methacrylic acid-methyl methacrylatecopolymers, and methacrylic acid-methyl methylacrylate copolymers. Dyestuffs or pigments can be added to the tablets or dragee coatings, forexample, for identification to characterize combinations of activecompound doses.

Other pharmaceutical preparations that can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules thatmay be mixed with fillers, such as lactose, binders, such as starches,and/or lubricants, such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, one or more active ingredients (e.g.,CoQ₁₀) can be dissolved or suspended in suitable liquids, such as fattyoils or liquid paraffin.

Examples of formulations for parenteral administration can includeaqueous solutions of one or more active ingredients in water-solubleform, for example, water-soluble salts, and alkaline solutions. Examplesof salts can include maleate, fumarate, succinate, S,S tartrate, or R,Rtartrate. In addition, suspensions of one or more of the activeingredients as oily injection suspensions can be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides or polyethylene glycol-400. Aqueous injection suspensionscan contain substances that increase the viscosity of the suspension,sodium carboxymethyl cellulose, sorbitol, and/or dextran.

The therapeutically effective amounts of the ubiquinone (e.g., CoQ₁₀)and a hypolipidemic agent can be administered to a subject on a desireddosing schedule. For example, a therapeutically effective amount of apharmaceutical composition comprising CoQ₁₀ can be administered aboutfour times daily, about three times daily, about twice daily, aboutdaily, about every other day, about three times weekly, about twiceweekly, about weekly, about every two weeks, or less often (as desired).In one example, a therapeutically effective amount of a pharmaceuticalcomposition includes 30-1,200 milligrams of CoQ₁₀ taken orally individed doses. In another example CoQ₁₀ can be administeredintravenously at a dose of around 5 mg/kg of body weight.

A therapeutically effective amount of the ubiquinone (e.g., CoQ₁₀)and/or a hypolipidemic agent can also be administered for a durationsufficient to provide a prophylactic effect. For example, atherapeutically effective amount of CoQ₁₀ can be administered daily forone year, for about six months, about a year, about two years, aboutfive years, about 10 years, or indefinitely. It will be apparent tothose of skill in the art that the dose, dosing schedule, and durationcan be adjusted for the needs of a particular subject, taking intoconsideration the subject's age, weight, severity of disease, and otherco-morbid conditions.

Toxicity and therapeutic efficacy of compositions comprising ubiquinoneand/or a hypolipidemic agent for use in the invention can be determinedusing standard pharmaceutical procedures in cell culture or experimentalanimals for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.

The following example is for the purpose of illustration only and is notintended to limit the scope of the claims, which are appended hereto.

Example 1 Materials and Methods LAD Ligation/Reperfusion andQuantification of Area at Risk and Infarct Size

All animal protocols were approved by the Animal Research Committee, andall animals were housed in the Association for Assessment andAccreditation of Laboratory Animal Care International-approved animalfacility of the Cleveland Clinic. Anterior wall MI (AMI) was performedas recently described (Askari, A. T. et al., J. Exp. Med. 197:615-624,2003). Briefly, AMI was induced in eight 20- to 25-g male littermatewild-type (C57BL/6J), ApoA1 heterozygote (ApoA1^(+/−)) or knockout(ApoA1^(−/−)) mice by ligation of the LAD 7-0 Prolene. Blanching anddysfunction of the anterior wall verified LAD ligation. After 30 minutesof LAD ligation, microsurgical scissors were used to cut the knot in theligature at the level of the myocardium, and animals subsequentlyunderwent reperfusion for 3 hours. Successful reperfusion was verifiedby return of red color to the tissue that was initially blanched at thetime of LAD ligation, as well as gross evidence of some recovery ofanterior wall motion Animals were kept on ventilator for the entire 3hours of reperfusion and subsequently analyzed for area at risk andinfarct size using 1% solution of 2,3,5-triphenyltetrazolium chloride(TTC) at 37° C. Briefly, once the LAD was ligated again, Evan's blue dye(1 mg/mL) was infused to define the volume of myocardium not at risk.After Evan's blue dye infusion, the heart was harvested and sectionedinto 3 pieces defined as the base, mid, and apex. The sections wereincubated in TTC solution for 15 minutes, rinsed, and then placed informalin overnight.

Determination of Reactive Oxygen Species (ROS) Production In Vivo

ROS production was assessed using in vivo hydroethidine dye (Kondo, T.et al., J. Neurosci. 17:4180-4189, 1997; Murakami, K. et al., J.Neurosci. 18:205-213, 1998), as previously described (Manabe, Y. et al.,Ann. Neurol. 55:668-675, 2004). Hydroethidine, a cell-permeableoxidative fluorescent dye, is oxidized to ethidium by superoxide(Carter, W. O. et al., J. Leukoc. Biol. 55:253-258, 1994; Bindokas, V.P. et al., J. Neurosci. 16:1324-1336, 1996). Ethidium, which exhibitspeak absorbance at 520 nm and an emission maximum at 600 nm, is trappedintracellularly by intercalating with DNA (Rothe, G. et al., MethodsEnzymol. 233:539-548, 1994). The fluorescence signal attributable toethidium reflects cumulative ROS production during the period betweenadministration of hydroethidine and killing of the animal. Hydroethidine(10 mg/kg) was injected into the jugular vein of anesthetized andpreviously infracted animal as described above and allowed to circulatefor 4 h. Mice were killed, and hearts were removed andparaffin-embedded. Serial sections (n=5) were cut and collected at 600μm intervals, and viewed with a confocal microscope. The analysis of ROSproduction was performed in a blinded manner by a differentinvestigator. Five randomly selected areas within the infarct zone wereselected and analyzed. Fluorescence intensity was measured in fiveserial sections per animal. The sum of the fluorescence intensity foreach region was divided by the total number of pixels analyzed andexpressed as relative fluorescence units.

TUNEL Assay

Heart sections were used to perform TUNEL staining with the In Situ CellDeath Detection kit (Roche Applied Science) per the manufacturer'sinstructions. Hearts were collected after 30 minutes ischemia/3 hreperfusion for assessment of TUNEL. Heart sections were incubated withTUNEL staining (Roche) for cell death and co-staining was performedusing DAPI and cells were visualized with a confocal microscope.TUNEL-positive-staining cells were counted at 40× magnification in 5randomly selected areas within the infarct zone and expressed aspositive cells per mm² and then compared between WT and ApoAI KO mice.At least 10 sections were analyzed throughout the entire longitudinalaxis of the hearts (n=5 hearts per group).

Mitochondrial Techniques

Three mouse hearts were pooled for isolation of cardiac mitochondria.Hearts were finely minced, placed in Chappell-Perry (CP 1) buffer (inmM: 100 KCl, 50 Mops, 5MgSO₄, 1 EGTA, 1 ATP), trypsin added (1 mg/g wetweight), and homogenized with a polytron tissue processor (BrinkmannInstruments, Westbury, N.Y.) for 2.5 s at a rheostat setting of 3.5. Thepolytron homogenate was incubated in homogenization tube for 10 minuteswith stirring at 4° C. CP 2 buffer (CP 1 with 2% fatty-acid free BSA),was added in the homogenate right after the incubation to stop thetrypsin digestion. Additional mixing and homogenization was performedusing 2 strokes with the loose pestle and 2 strokes with the tightpestle. Then, the homogenate was centrifuges at 500×g for 10 minutes.The supernatant was saved for isolation of mitochondria and the palletwas washed twice (centrifuge at 3000×g) and then resuspended in KME (inmM: 100 KCl, 50 MOPS, and 0.5 EGTA). Mitochondrial protein concentrationwas measured by the Lowry method, using bovine serum albumin as astandard.

Oxygen consumption in mitochondria was measured using a Clark-typeoxygen electrode at 30° C. Mitochondria were incubated in a solutionincluding 80 mM KCl, 50 mM MOPS, 1 mM EGTA, 5 mM KH₂PO₄ and 1 mgdefatted, dialyzed BSA/ml at pH 7.4. Glutamate (complex I substrate, 20mM) plus malate (2 mM), succinate (complex II substrate, 20 mM) plusrotenone (7.5 mM), and N,N,N′,N′-tetramethyl p-phenylenediamine(TMPD)-ascorbate (complex IV substrate, 10 mM) plus rotenone (7.5 mM),were used. State 3 (ADP-stimulated), state 4 (ADP-limited) respiration,respiratory control ratios, the ADP/O ratio, and dinitrophenol-uncoupledrespiration were determined Endogenous substrates were depleted byaddition of 0.1 mM ADP before glutamate stimulated respiration.

The following enzyme activities were measured in detergent-solubilizedmitochondria using previously described methods (Hoppel, C. L. et al.,J. Clin. Invest. 80:71-77, 1987; Lesnefsky, E. J. et al., Am. J.Physiol. 273:H1544-H1554, 1997): NADH-cytochrome c reductase (NCR,rotenone sensitive); succinate cytochrome c reductase (SCR)-antimycin Asensitive; complex II, thermoyltrifluoroacetone (TTFA), sensitive;complex III, antimycin A sensitive, and citrate synthase (CS).

Net H₂O₂ production from mitochondria was measured using the oxidationof fluorogenic indicator amplex red in the presence of horseradishperoxidase. Amplex red assay was obtained from Molecular Probes (Eugene,Oreg.). Glutamate and succinate were used as complex I and complex IIsubstrates, and the concentration of substrates is the same as that usedto measure oxidative phosphorylation (Chen, Q. et al., J. Biol. Chem.278:36027-36031, 2003.

Statistical Analyses

All data are expressed as mean±SD. Statistical analysis was performedwith use of SPSS software (version 10.0 for Windows, SPSS Inc).Comparisons between two groups were statistically evaluated by Student'st-test. A value of P≦0.05 was considered statistically significant.

Results Effect of Genetic Background on Infarct Size in Mice

Due to increased mortality rate in ApoAI KO mice after chronic ligationof the proximal left anterior descending artery (LAD) (˜80-90% withinthe first 24 hrs), acute myocardial infarction was achieved by inducing30 min of LAD ischemia and subsequent reperfusion for 3 hours. Mice werekept on ventilator for the entire experiment. There were no differencesin the area at risk following LAD ligation between WT, ApoA-I+/− andApoA-I−/− mice following LAD ligation, 49.9±11.2%, 49.7±3.2% and51.7±4.4%, respectively. Conversely the infarct size as a percent of theAAR (IS/% AAR) correlated with the level of ApoA-1 with the largestinfarcts seen in the ApoA-I null mice compared to ApoA-I het and WT mice(25.3±7.8%, n=4 vs. 17.8±3.0%, n=6 and 13.1±2.8%, n=4, respectively,FIG. 1), WT vs. ApoA-I het p=0.042; WT vs. ApoA-I null, p=0.002).

In Situ Detection of ROS

We postulated that the increase in infarct size in the ApoAI KO micecould be due to the increased production of ROS since ROS plays a majorpathogenic role in ischemic injury. We used hydroethidine technique toquantify ROS release in WT and APOAI null mice after reperfusion(Representative images are shown in FIGS. 2A-B). There was a trend foran increase in mean fluorescence intensity in ApoAI null mice comparedto WT mice (87.9±47 and 54.6±17.9, respectively, p=0.23, FIG. 2C).

In Situ DNA Fragmentation by TUNEL Staining

To determine if there was increased apoptosis is responsible for theobserved injury in ApoAI KO mice, the TUNEL method was employed todetect apoptotic nuclei in myocardial cells. Quantitatively, the numberof TUNEL positive cells/mm² trended higher in the ApoAI null micecompared to WT mice (19.7±13.5 and 12.1±11.1, respectively, p=0.17);however, this increase was not statistically significant.

Mitochondrial Oxidative Phosphorylation

Mitochondrial oxidative metabolism was measured with glutamate,succinate and TMPD-ascorbate as substrates. Oxygen consumption underADP-stimulated (state 3), ADP-limited (state 4) conditions are shown inTable 1.

TABLE 1 Oxidative phosphorylation ApoA1KO (n = 4) WT (n = 5) 2 mM 2 mMSubstrate State 3 State 4 RCR ADP/O ADP State 3 State 4 RCR ADP/O ADPGlutamate 288 ± 1.9    37 ± 3.4  8.3 ± 0.9 3.2 ± 0.1 345 ± 40  298 ±17.9 38 ± 4  8 ± 0.4 3.3 ± 0.1 341 ± 44.7 Pyruvate +  437 ± 16.2 59.6 ±5.6  7.4 ± 0.5 3.6 ± 0.1 489.3 ± 32    416 ± 35.5   60 ± 14.7  7 ± 1 3.7± 0.3 454 ± Malate 40.7 Succinate 575 ± 10* 193 ± 10  3 ± 0*§ 1.6 ± 0.1555 ± 40 674 ± 29  193 ± 12  3.5 ± 0.1*§ 1.6 ± 0.1 656 ± 28 DHQ 663 ±30  217 ± 50  3.1 ± 0.5 1.6 ± 0.1 791 ± 48 570 ± 125 184 ± 19  3 ± 0.31.5 ± 0.1 648 ± 72 TMPD 1191 ± 65  1918 ± 134 206 ± 57 1712 ± 99  1222 ±52  1818 ± 70  246 ± 24 15.72 ± 55   Values are means ± SE. Oxidativephosphorylation of glutamate in nanoatoms O min⁻¹ mg protein⁻¹. RCR,respiratory control rate; ADP/O, ADP-to-O ratio. *P < 0.05 vs.corresponding WT. §P < 0.005.

State 3 respiration, state 4 respiration, respiration control ratio(RCR), and the ADP/O ratio were similar in WT and KO mice when glutamatewas used as the substrate (Table 1). With succinate (plus rotenone), asa complex II substrate, a decrease in both state 3 respiration, anduncoupled respiration occurred in KO mice. The decreased coupling ofrespiration observed in KO mice indicated by the decrease in RCR wasmainly due to a decreased state 3 respiration, rather than an increasedstate 4 respiration or phosphorylation apparatus defect since state 4respiration was not altered in KO mice. Furthermore,dinitrophenol-uncoupled respiration was decreased in KO mice, localizingthe respiratory defect to the electron transport chain (ETC).

Substrates that donate electrons to specific sites in the ETC were usedunder conditions of maximal ADP-stimulated respiration to identify sitesof damage to the ETC. The maximal ADP-stimulated respiration measuredusing 2 mM ADP decreased in KO mice with succinate as substrate whenrespiration supported by electron flow from complex II, ubiquinone,complex III, cytochrome c and complex IV. In contrast, the maximalADP-stimulated respiration was not affected in both WT and KO mice withglutamate, DHQ and TMPD-ascorbate as substrates when electron flowrespectively from complex I, ubiquinone, complex III to final cytochromec and complex IV.

ETC Enzyme Activities

NADH cytochrome c reductase, measures of the activity of complex I andIII was similar in both KO and WT mice (FIG. 3A). The activity ofsuccinate cytochrome c reductase (SCR, antimycin A sensitive) wasmarkedly decreased in KO mice, localizing a defect to complex II,ubiquinone and complex III of the ETC (Table 2).

TABLE 2 Enzyme activities Enzyme ApoAI KO (n = 4) WT (n = 5) Complex I865 ± 71 810 ± 59 NCR 5120 ± 506 5612 ± 488 Complex III 5654 ± 580 6413± 449 SCR  334 ± 28* 764 ± 50 Complex II 759 ± 75 789 ± 57 CitrateSynthase 3809 ± 166 4036 ± 78  Values are mean ± SE. *P < 0.0001 vs.corresponding WT.

However, the activity of complex III was not changed compared to WT miceand the activity of complex II was surprisingly normal (FIG. 3A). Thus,the defect in ETC of KO mice was likely at the Q pool, which altered theelectron transfer from complex II to complex III. The activity ofcitrate synthase, a mitochondrial matrix marker enzyme, was unaltered inboth WT and KO mice (FIG. 3B).

H₂O Production in Mitochondria

Since horseradish peroxidase and amplex red do not enter intactmitochondria, only H₂O₂ that is released from mitochondria (net releaseof H₂O₂, pmol/mg/30 min) is detected by this assay. The net productionof H₂O₂ in WT and KO mice was similar with glutamate as complex Isubstrate, as well as with succinate plus rotenone as a complex IIsubstrate (Table 3).

TABLE 3 Effect of genetic background of mice on mitochondrial H₂O₂production ApoAI KO WT (pmol/mg/30 min.) (pmol/mg/30 min.) Complex Isubstrate Glutamate/malate 570 ± 73 872 ± 141 Glutamate/malate plus 271± 58 425 ± 66  rotenone Complex II substrate Succinate  686 ± 117 923 ±119 Succinate plus rotenone 207 ± 79 371 ± 8  Means ± SEM.Concentrations used: glutamate 10 mM, malate 2.5 mM, succinate 5 mM.When succinate was used as a substrate, rotenone (2.4 μm) was added.

Effect of CoQ10 on Infarct Size in ApoAI Null Mice

The analyses of mitochondrial function in the WT and ApoAI null micesuggested that there is a defect in the mitochondria of ApoAI null micethat leads to decreased flux in the succinate cytochrome c reductasepathway secondary to a deficiency in the Q pool. To determine if the Qpool was then a target for reversing the adverse effects of ApoAIdeficiency, we inject CoQ10 ip (1 mg/kg/day) into WT and ApoAI null micefor 3 days prior to inducing ischemia reperfusion. The data in FIGS.3A-B demonstrate that the administration of CoQ10 to ApoAI null micecompletely corrected the defect seen in ApoAI null mice leading to >100%decrease in the infarct size as a percent area at risk (FIG. 4). Therewas a non-significant trend (p=0.15) towards a decrease in IS/% AAR inthe WT mice treated with CoQ10 compared to WT controls.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes, and modifications are within the skill of the artand are intended to be covered by the appended claims.

1. A method for predicting myocardial damage in a subject having or atrisk of cardiac disease, the method comprising: determining a level ofapolipoprotein AI (ApoAI) in the subject; determining a level ofCoenzyme Q₁₀ (CoQ₁₀) in the subject; comparing the determined levels ofApoA1 and CoQ₁₀ to control levels, wherein a decreased level of ApoAIand a decreased level of CoQ₁₀ compared to control levels are indicativeof the subject having an increased risk of greater myocardial damagefollowing a myocardial infarction.
 2. The method of claim 1, furthercomprising the step of obtaining one or more bodily samples from thesubject, the one or more bodily samples including CoQ₁₀ and ApoAI. 3.The method of claim 2, the one or more bodily samples comprising plasma.4. The method of claim 1, wherein the level of ApoAI in the subject isdetermined using an ELISA assay.
 5. The method of claim 1, wherein thelevel of CoQ₁₀ in the subject is determined using high-performanceliquid chromatography.
 6. The method of claim 1, wherein the myocardialdamage comprises a ratio of an area of ischemic myocardial tissue tototal area of myocardial tissue.
 7. The method of claim 1, the controllevels include normal levels of ApoA1 and CoQ10 in a population ofapparently healthy subjects.
 8. The method of claim 1, wherein thecardiac disease is selected from the group consisting of myocardialinfarction, coronary artery disease, angina, atherosclerosis, aneurysm,congestive heart failure, left ventricular dysfunction, cerebrovasculardisease, and cerebrovascular accident.
 9. A method of determiningincreased risk of greater myocardial damage in a subject having or atrisk of cardiac disease, the method comprising: determining a level ofapolipoprotein AI (ApoAI) in the subject; determining a level ofCoenzyme Q₁₀ (CoQ₁₀) in the subject; comparing the determined levels ofApoA1 and CoQ₁₀ to control levels, wherein a decreased level of ApoAIand a decreased level of CoQ₁₀ compared to control levels are indicativeof the subject having an increased risk of greater myocardial damagefollowing a myocardial infarction.
 10. The method of claim 9, furthercomprising the step of obtaining one or more bodily samples from thesubject, the one or more bodily samples including CoQ₁₀ and ApoAI. 11.The method of claim 10, the one or more bodily samples comprisingplasma.
 12. The method of claim 9, wherein the level of ApoAI in thesubject is determined using an ELISA assay.
 13. The method of claim 9,wherein the level of CoQ₁₀ in the subject is determined usinghigh-performance liquid chromatography.
 14. The method of claim 9,wherein the myocardial damage comprises a ratio of an area of ischemicmyocardial tissue to total area of myocardial tissue.
 15. The method ofclaim 9, the control levels include normal levels of ApoA1 and CoQ10 ina population of apparently healthy subjects.
 16. The method of claim 9,wherein the cardiac disease is selected from the group consisting ofmyocardial infarction, coronary artery disease, angina, atherosclerosis,aneurysm, congestive heart failure, left ventricular dysfunction,cerebrovascular disease, and cerebrovascular accident.
 17. A method ofdetermining increased risk of greater myocardial damage in a subjecthaving or at risk of cardiac disease, the method comprising: determininga level of HDL in the subject; determining a level of Coenzyme Q₁₀(CoQ₁₀) in the subject; comparing the determined levels of ApoA1 andCoQ₁₀ to control levels, wherein a decreased level of ApoAI and adecreased level of CoQ₁₀ compared to control levels are indicative ofthe subject having an increased risk of greater myocardial damagefollowing a myocardial infarction.
 18. The method of claim 17, furthercomprising the step of obtaining one or more bodily samples from thesubject, the one or more bodily samples including CoQ₁₀ and HDL.
 19. Themethod of claim 18, the one or more bodily samples comprising plasma.20. The method of claim 17, wherein the level of HDL in the subject isdetermined using a fluorometric assay.
 21. The method of claim 17,wherein the level of CoQ₁₀ in the subject is determined usinghigh-performance liquid chromatography.
 22. The method of claim 17,wherein the myocardial damage comprises a ratio of an area of ischemicmyocardial tissue to total area of myocardial tissue.
 23. The method ofclaim 17, the control levels include normal levels of HDL and CoQ10 in apopulation of apparently healthy subjects.
 24. The method of claim 17,wherein the cardiac disease is selected from the group consisting ofmyocardial infarction, coronary artery disease, angina, atherosclerosis,aneurysm, congestive heart failure, left ventricular dysfunction,cerebrovascular disease, and cerebrovascular accident. 25-33. (canceled)